bis(2-ethylhexyl) (4-hydroxy-3,5-dimethoxybenzyl)malonate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2007-10-24 to 2007-12-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

adopted 23 March 2006

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

29 December 1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Remarks:

As the water solubility of the test substance is <0.02 mg/L and no effects on algae growth was observed in a pretest up to the limit concentration (nominal 100 mg/L), no analytical monitoring was performed during this study.

Details on sampling:

NA

Vehicle:

no

Details on test solutions:

The test medium (reconstituted water as vehivle and test material) was freshly prepared.
The calibrated flask with test material and vehicle, reconstituted water, was treated in an ultrasound bath for 1 hour.
Subsequently, the preparation was stirred with a magnetic stirrer for further 23 hours.
Afterwards, the preparation was filtered through a filter funnel with a pore size of 10-16 µm.
The filtrate was used for the study.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

The test system used for this study was a green unicellular algae species, Desmodesmus subspicatus, strain No. SAG 86.81, supplied by the "Sammlung von Algenkulturen, Pflanzenphysiologisches Institut der Universitat Gottingen". This strain is recommended by international guidelines.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

250 mg CaCO3

Test temperature:

23-24°C

pH:

Concentration pH without algae pH with algae
([mg/L] Start End Start End
0 7.87 7.85 7.87 10.14
100 7.78 7.87 7.78 10.15

Dissolved oxygen:

80%

Salinity:

for details see composition of reconstituted water "details on test conditions"

Nominal and measured concentrations:

The solution of a nominal test material concentration of 100 mg/L was tested in an open static system.

Details on test conditions:

The study was located in an air-conditioned room. Preparations of 100 mL each were continuously shaken in 300 mL Erlenmeyer flasks by a pulsating panel. The flasks were completely sealed with sterilized stoppers. The flasks were continuously illuminated for the exposure period by fluorescent tubes installed above the panel containing the flasks.

The test medium (reconstituted water and test material) was freshly prepared. The calibrated flask with test material and vehicle, reconstituted water, was treated in an ultrasound bath for 1 hour. Subsequently, the preparation was stirred with a magnetic stirrer for further 23 hours. Afterwards, the formulation was given to a separating funnel for 6 hours. The middle phase of the preparation was filtered through a filter funnel with a pore size of 10 - 16 um. The filtrate was used for the study.

Volumes of 100 mL reconstituted water or test medium (reconstituted water with test material) were freshly prepared before the algae suspensions were added. The control and the test medium were placed into 300 mL Erlenmeyer flasks and covered with sterilized cellulose stoppers.

The flasks were incubated under standardized conditions, in an air-conditioned room under continuous rotating and continuous illumination. The light intensity was approx. 7500 to 9500 Lux. The illumination was achieved by fluorescent tubes (Philips Master tubes TL5 80W/840 HO) instaIled above a rotating panel with the flasks.

The experimental part was started by inoculation of algae to the different groups so that the flasks contained about 10,000 cells/mL. The algae were taken from an exponentially growing preculture (growth rate 120.7) which was set up 3 days prior to the experimental part under the same conditions as in the final study.

TEST SYSTEM
- Test vessel: 300 mL Erlenmeyer flasks
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 100 mL
- Initial cells density: 10,000 cells/mL
- Control end cells density: 1,061,451 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes
- Details on composition of the reconstituted water:
Macro-nutrients:
50.00 mg/L NaHCO3
18.00 mg/L CaCl2 x 2 H2O
15.00 mg/L NH4Cl
15.00 mg/L MgSO4 x 7 H2O
12.00 mg/L MgCl2 x 6 H2O
1.60 mg/L KH2PO4

Trace elements:
100.00 µg/L Na2EDTA x 2 H2O
80.00 µg/L FeCl3 x 6 H2O
415.00 µg/L MnCl2 x 4 H2O
185.00 µg/L H3BO3
7.00 µg/L Na2MoO4 x 2 H2O
3.00 µg/L ZnCl2
1.50 µg/L CoCl2 x 6 H2O
0.01 µg/L CuCl2 x 2 H2O

The pH of the reconstituted water after aeration is approximately 8.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuously
- Light intensity and quality: 7500 to 9500 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter

TEST CONCENTRATIONS
- Justification for using less concentrations than requested by guideline: Limit test
- Range finding study: yes
- Test concentrations: 100 mg/L (nominal)

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

Pretest
In a pretest under static conditions, inhibition of the growth rate of the algae, Desmodesmus subspicatus, was not observed at a concentration of nominally 100 mg/L.

Main study
In the control group, the factor of cell growth was approx. 106.
The algae increased from 10,000 cells/mL at the start to 1,061,451 cells/mL after 72 hours. For more details on the results see attachment.

see attached table

Validity criteria fulfilled:

yes

Conclusions:

An aqueous preparation of nominal 100 mg/L of the test item revealed no toxic effect in the test system. Therefore, the 72 hours EC50 could not be determined, because it exceeded the maximum solubility of the test material in reconstituted water (analytical EC50> 0.02 mg/L).

Executive summary:

Purpose
The purpose of this assay was to identify the aquatic toxicity potential of the test substance in algea to provide a rational basis for hazard estimation for the test item in aquatic environments.

Study Design
The influence of the test material on the growth and growth rate of the green algae species Desmodesmus subspicatus was tested.
The study design included one control and one test material group with three replicates, each containing 100 mL reconstituted water or test medium and about 10,000 cells/mL at the start of the experimental phase.
The study was performed as a limit test in an open static test system. The algae were exposed to a filtrate of nominal 100 mg/L.
The growth of the algae was calculated after 24, 48, and 72 hours exposure in the test medium.

Results

The analysis of the saturated aqueous preparation revealed that the water solubility of the test item in reconstituted water was < 0.02 mg/L. An exact value could not be determined. The test material concentration in the aqueous medium could not be quantified since it was below the limit of quantification.

After exposure of Desmodesmus subspicatus for 72 hours the following results were obtained:

 

	Mean cell density per mL					Growth inhibition (0-72 h)
Nom. mg/L		0 hour	24 hours	48 hours	72 hours	IA	Iµt
0		10000	62388	236833	1061451
100		10000	68161	244403	1388583
	IA		-11,0	-4,8	-26,2	-22,0	-5,8

I_A= inhibition of growth in percent
I_µt= inhibition of growth rate in percent
Nom. = nominal concentration

For the test material, the following ECS0 values and no effect concentration for Desmodesmus subspicatus were determined:

72 h E_bC₅₀> 0.02 mg/L (nominal >100 mg/L)
72 h E_rC₅₀  > 0.02 mg/L (nominal >100 mg/L)
NOEC >  0.02 mg/L (nominal >100 mg/L)

Conclusion
An aqueous preparation of nominal 100 mg/L of the test substance revealed no toxic effect in the test system. Therefore, the 72 hours EC50 could not be determined, because it exceeded the maximum solubility of the test material in reconstituted water (EC50> 0.02 mg/L).

Description of key information

An aqueous preparation of nominal 100 mg/L of the test substance revealed no toxic effect in the test system. Therefore, the 72 hours EC50 could not be determined, because it exceeded the maximum solubility of the test material in reconstituted water (EC50> 0.02 mg/L).

Additional information

Purpose
The purpose of this assay was to identify the aquatic toxicity potential of the test substance in algea to provide a rational basis for hazard estimation for the test item in aquatic environments.

Study Design
The influence of the test material on the growth and growth rate of the green algae species Desmodesmus subspicatus was tested.
The study design included one control and one test material group with three replicates, each containing 100 mL reconstituted water or test medium and about 10,000 cells/mL at the start of the experimental phase.
The study was performed as a limit test in an open static test system. The algae were exposed to a filtrate of nominal 100 mg/L.
The growth of the algae was calculated after 24, 48, and 72 hours exposure in the test medium.

Results

The analysis of the saturated aqueous preparation revealed that the water solubility of the test item in reconstituted water was < 0.02 mg/L. An exact value could not be determined. The test material concentration in the aqueous medium could not be quantified since it was below the limit of quantification.

After exposure of Desmodesmus subspicatus for 72 hours the following results were obtained:

 

	Mean cell density per mL					Growth inhibition (0-72 h)
Nom. mg/L		0 hour	24 hours	48 hours	72 hours	IA	Iµt
0		10000	62388	236833	1061451
100		10000	68161	244403	1388583
	IA		-11,0	-4,8	-26,2	-22,0	-5,8

I_A= inhibition of growth in percent
I_µt= inhibition of growth rate in percent
Nom. = nominal concentration

For the test material, the following ECS0 values and no effect concentration for Desmodesmus subspicatus were determined:

72 h E_bC₅₀> 0.02 mg/L (nominal >100 mg/L)
72 h E_rC₅₀  > 0.02 mg/L (nominal >100 mg/L)
NOEC >  0.02 mg/L (nominal >100 mg/L)

Conclusion
An aqueous preparation of nominal 100 mg/L of the test substance revealed no toxic effect in the test system. Therefore, the 72 hours EC50 could not be determined, because it exceeded the maximum solubility of the test material in reconstituted water (EC50> 0.02 mg/L).